This invention pertains to the transformation, on the one hand, and organogenic regeneration, on the other hand, of cotton plants, a commercially important crop. Both nuclear and plastid transformation are embraced.
Cotton has been traditionally recalcitrant to regeneration in vitro. Most regeneration successes have entailed the sole use of Coker lines which respond in. tissue culture but are not agronomically important (Chlan et al., 1995; Firoozabady et al., 1987; Peeters et al., 1994; Shoemaker et al., 1986; Umbeck et al., 1987). Most (if not all) developed regeneration protocols entail the production of embryogenic callus from seedling explants such as cotyledon and hypocotyl sections, followed by the formation of somatic embryos with subsequent germination and conversion into mature cotton plants (Firoozabady and DeBoer, 1993; Firoozabady et al., 1987; Peeters et al., 1994; Rajasekaran et al., 1996; Shoemaker et al., 1986; Umbeck et al., 1987; U.S. Pat. Nos. 5,159,135, and 5,244,802). This type of regeneration procedure could take up to 40 weeks and could produce unwanted mutations due to the presence of a prolonged callus phase prior to regeneration.
Cotton tissues have been successfully transformed with A. tumefaciens prior to generation of embryogenic callus used in regeneration (Firoozabady et al., 1987; Rajasekaran et al., 1996; Umbeck et al., 1987). Cotton has also been transformed via biolistics with tissues also undergoing regeneration via somatic embryogenesis (Rajasekaran et al., 1996). A few protocols have recently utilized intact meristem-tips as targets in biolistics-based transformations with regeneration occurring via a more direct organogenic route (Chlan et al., 1995; Finer and McMullen, 1990; McCabe and Marinelli, 1993). Although this has overcome some regeneration obstacles, it is technically demanding. Due to their extremely small size ( less than 1.0 mm), the meristem-tips have to be excised with the aid of a dissecting microscope and once isolated, need to be utilized shortly thereafter.
Accordingly, it remains an object of those of skill in the art to develop a method for regeneration of cotton, in vitro, with an eye to obtaining genetic variation providing desirable qualities. In particular, it is an object of those of skill in the art to obtain a method for transforming plant tissues with exogenous DNA, or obtaining mutations of endogenous DNA, and regenerating the tissues containing these DNA alterations and additions into mature, fertile plants.